The objective of this project is to develop an adenoviral vector vaccine against HIV that is effective in stimulating cell-mediated immunity (CMI) in Ad-immune animals. The HIV vaccine will be used to protect against infection and to treat the infected. HIV Gag, Pol, and Nef have been reported to be useful for vaccine development. Evidence indicates that a broad CMI response is needed to treat or prevent HIV infection. Adenovirus (Ad) vector vaccines induce CMI responses and have emerged as a leading candidate to be used as a vaccine delivery platform. First Generation Ad vaccines have proven less effective than anticipated and adverse reactions are in question. Furthermore, pre-existing Ad immunity of most humans causes decreased effectiveness. To address these issues, we have developed an advanced Ad based vector that is devoid of early genes E1, E3, and E2B. These "E2B-deleted" vectors, with deletions in the polymerase and preterminal protein genes, have an expanded cloning capacity and greatly reduced expression of viral late genes as compared to First Generation. The reduced expression of multiple Ad viral genes has been demonstrated to be advantageous for vaccine development for reasons such as reduced antigenic competition, greater longevity of transgene expression, which provides increased immunologic stimulus and reduced adverse effects. Such advantages are important in the presence of pre-existing Ad immunity, and provide the E2B Ad vectors stealth-like attributes. The Company has exclusive license for the new Ad vector system and the E.C7 cell line that supports vector production. The proposed studies are designed to construct and test the effectiveness of HIV vaccines based on the new E2B-deleted Ad vector platform, which carry Gag gene. Ad vaccines have been tested for their potential to induce HIV memory CMI responses as a prime and for their re-immunization (boost) potential in Ad-immune cynomolgus macaques. The phenotypes of HIV-1 Gag-specific T-cells, epitopes recognized, and polyfunctional T- cells secreting multiple cytokines (IL-2, IFN-y, TNF-a, MIP1-[unreadable], and IL-10) will be evaluated to compare the difference in CMI response induced by Ad5-E2b-gag and Ad5-FG-gag vaccine in Ad-immune macaques, which may give insight into the reason why Ad5-E2b-gag can overcome the barrier of pre-existing Ad immunity. This project will elucidate the CMI repertoire of the responses by macaques following our vaccine immunization. Our goal is to initiate a Phase I clinical trial using an Ad vector platform within two years of funding. PUBLIC HEALTH RELEVANCE: The overall goal of this project is to compare the immunologic responses of vaccination using first generation Ad5-FG-gag and our novel second-generation Ad5-E2b-gag vaccine in cynomolgus macaques that have been previously immunized against adenovirus (Ad) in order to develop an effective, safe, easy to produce, stable, and easy to use HIV vaccine.